Bottom Line:
When DOG-1 is absent but crosslinking agents are present the G-rich structures are readily covalently crosslinked, resulting in increased crosslinks formation and thus giving increased crosslink sensitivity.In this interpretation DOG-1 is neither upstream nor downstream in the FA pathway, but works alongside it to limit the availability of crosslink substrates.This model reconciles the crosslink sensitivity observed in the absence of DOG-1 function with its unique role in maintaining G-Rich DNA and will help to formulate experiments to test this hypothesis.

ABSTRACTC. elegans provides an excellent model system for the study of the Fanconi Anemia (FA), one of the hallmarks of which is sensitivity to interstrand crosslinking agents. Central to our understanding of FA has been the investigation of DOG-1, the functional ortholog of the deadbox helicase FANCJ. Here we review the current understanding of the unique role of DOG-1 in maintaining stability of G-rich DNA in C. elegans and explore the question of why DOG-1 animals are crosslink sensitive. We propose a dynamic model in which noncovalently linked G-rich structures form and un-form in the presence of DOG-1. When DOG-1 is absent but crosslinking agents are present the G-rich structures are readily covalently crosslinked, resulting in increased crosslinks formation and thus giving increased crosslink sensitivity. In this interpretation DOG-1 is neither upstream nor downstream in the FA pathway, but works alongside it to limit the availability of crosslink substrates. This model reconciles the crosslink sensitivity observed in the absence of DOG-1 function with its unique role in maintaining G-Rich DNA and will help to formulate experiments to test this hypothesis.

fig1: Schematic of the dog-1-derived translocation, hT4 (V; X). Sequences near the left end of chromosome V were deleted (red box), whereas the right end of chromosome X was duplicated (blue box). PCR primers were designed and used to determine the DNA sequence across the junction of V and X in hT4 (described in [10]).

Mentions:
Further work from our laboratory revealed that in the absence of DOG-1 large chromosomal rearrangements occurred [10]. The rearrangements included larger deletions, duplications of chromosomal fragments, and translocations between chromosomes, in addition to the small deletions detectable by PCR. These large rearrangements were identified because they acquired lethal mutations, which could be isolated and characterized with the use of a balancer chromosome that provided a rescuing wild-type allele in a stable genetic construct (reviewed in [13]). The analysis showed that 1% of the chromosomes acquired lethal lesions [10], giving a forward mutation frequency greater than tenfold of the spontaneous frequency. The frequency is equivalent to that for 500 Rads of ionizing radiation [14]. Rearrangements derived from dog-1 mutant that were examined by aCGH revealed that in most (but not all) cases the breakpoints occurred in G-rich DNA. In one example, a translocation between chromosome V and the X-chromosome was formed. In this case, the right end of the X-chromosome was duplicated and attached to the left breakpoint of a deletion at left end of chromosome V (Figure 1). The breakpoint on chromosome V is in a 24 bp G/C tract, while the breakpoint on the X is in a “short” 13 bp G-rich sequence. In vertebrates, large rearrangements have also been observed in the absence of FANCJ function. In avian DT40 cell lines, large-scale genomic deletions occurred at the rearranged immunoglobulin heavy chain locus (IgH) in the absence of FANCJ, but not other FA genes [15]. These researchers found that in FANCJ mutant cells cultured for two months, G4 sequences detected by aCGH were found at the breakpoints of one deletion. However, not all breaks occurred in G-rich DNA, suggesting that other sequences are also susceptible to breakage in the absence of FANCJ.

fig1: Schematic of the dog-1-derived translocation, hT4 (V; X). Sequences near the left end of chromosome V were deleted (red box), whereas the right end of chromosome X was duplicated (blue box). PCR primers were designed and used to determine the DNA sequence across the junction of V and X in hT4 (described in [10]).

Mentions:
Further work from our laboratory revealed that in the absence of DOG-1 large chromosomal rearrangements occurred [10]. The rearrangements included larger deletions, duplications of chromosomal fragments, and translocations between chromosomes, in addition to the small deletions detectable by PCR. These large rearrangements were identified because they acquired lethal mutations, which could be isolated and characterized with the use of a balancer chromosome that provided a rescuing wild-type allele in a stable genetic construct (reviewed in [13]). The analysis showed that 1% of the chromosomes acquired lethal lesions [10], giving a forward mutation frequency greater than tenfold of the spontaneous frequency. The frequency is equivalent to that for 500 Rads of ionizing radiation [14]. Rearrangements derived from dog-1 mutant that were examined by aCGH revealed that in most (but not all) cases the breakpoints occurred in G-rich DNA. In one example, a translocation between chromosome V and the X-chromosome was formed. In this case, the right end of the X-chromosome was duplicated and attached to the left breakpoint of a deletion at left end of chromosome V (Figure 1). The breakpoint on chromosome V is in a 24 bp G/C tract, while the breakpoint on the X is in a “short” 13 bp G-rich sequence. In vertebrates, large rearrangements have also been observed in the absence of FANCJ function. In avian DT40 cell lines, large-scale genomic deletions occurred at the rearranged immunoglobulin heavy chain locus (IgH) in the absence of FANCJ, but not other FA genes [15]. These researchers found that in FANCJ mutant cells cultured for two months, G4 sequences detected by aCGH were found at the breakpoints of one deletion. However, not all breaks occurred in G-rich DNA, suggesting that other sequences are also susceptible to breakage in the absence of FANCJ.

Bottom Line:
When DOG-1 is absent but crosslinking agents are present the G-rich structures are readily covalently crosslinked, resulting in increased crosslinks formation and thus giving increased crosslink sensitivity.In this interpretation DOG-1 is neither upstream nor downstream in the FA pathway, but works alongside it to limit the availability of crosslink substrates.This model reconciles the crosslink sensitivity observed in the absence of DOG-1 function with its unique role in maintaining G-Rich DNA and will help to formulate experiments to test this hypothesis.

ABSTRACTC. elegans provides an excellent model system for the study of the Fanconi Anemia (FA), one of the hallmarks of which is sensitivity to interstrand crosslinking agents. Central to our understanding of FA has been the investigation of DOG-1, the functional ortholog of the deadbox helicase FANCJ. Here we review the current understanding of the unique role of DOG-1 in maintaining stability of G-rich DNA in C. elegans and explore the question of why DOG-1 animals are crosslink sensitive. We propose a dynamic model in which noncovalently linked G-rich structures form and un-form in the presence of DOG-1. When DOG-1 is absent but crosslinking agents are present the G-rich structures are readily covalently crosslinked, resulting in increased crosslinks formation and thus giving increased crosslink sensitivity. In this interpretation DOG-1 is neither upstream nor downstream in the FA pathway, but works alongside it to limit the availability of crosslink substrates. This model reconciles the crosslink sensitivity observed in the absence of DOG-1 function with its unique role in maintaining G-Rich DNA and will help to formulate experiments to test this hypothesis.